We have found our new MALDI-ITMS instrument to be an enormously powerful tool for the analysis of proteins [Highlight #6 and 7]. The strategy of choice for many of these applications involves proteolytic digestion of the protein of interest followed by peptide mapping and MS/MS analysis of the individual proteolytic products. To obtain maximal information from the MS/MS spectra, it is necessary that we have as complete an understanding as possible of the systematics of fragmentation of MALDIproduced peptide ions in the ion trap mass spectrometer. We have already determined that tryptic peptides (and especially arginine-containing tryptic peptides) undergo highly preferential fragmentation adjacent to Glu and Asp residues (Qin, Chait, J. Am. Chem. Soc., 1995). It cannot be overemphasized how useful this finding has proved to be for the solution of challenging biological problems (e.g., protein identification). In addition to the preferential cleavage at acidic residues, we have made a number of other systematic observations (e.g., special cleavage patterns surrounding Pro residues, at terminal GIn residues, in multiple Arg-containing peptides, across disulfide bonds, at oxidized Met residues etc.). However, to put such observations on a fmn footing will require considerable additional studies. We, thus, propose to systematically study the MALDI-ITMS fragmentation of > 1000 additional peptides and carefully correlate their primary structures and mass with the observed fragmentation. In particular, we will separately investigate the fragmentation of peptides generated by digestion with respectively trypsin (to yield peptides with C-terminal Arg or Lys), V8 protease (to yield C-terminal Glu), proteinase AspN (to yield N-terminal Asp), and chymotrypsin (to yield C-terminal hydrophobic residues). When particularly interesting systematics are observed, we will on occasion synthesize peptide analogues to assess the generality of these observations or to more strictly define the structural features that are important for the observed fragmentation. We plan to incorporate all of our observed fragmentation information into our publicly available mass spectrometric database "MASSBANK" to provide the complete set of fragmentation spectra to the mass spectrometric community at large. Interested researchers will be in the position to deduce additional systematics of fragmentation behavior related to their own interests or to use the information as a leaming tool.